Project Summary/Abstract Clinical studies indicate that the aversive somatic states experienced during opioid withdrawal predict drug craving and increase the risk of relapse. However, little is known about the neural circuitry mediating these aversive states, or how that circuitry interacts with known components of the neural circuitry mediating relapse to opioid seeking. Recently, the paraventricular thalamic (PVT) projection to the nucleus accumbens (NAc) was implicated in opioid withdrawal symptoms in mice exposed to repeated non-contingent morphine. However, it is unknown whether withdrawal from heroin self-administration engages the PVT?NAc pathway to drive relapse by precipitating an aversive and/or painful state. To fill this gap in knowledge, during the K99 mentored phase of this proposal, I will receive training in optogenetics and brain slice electrophysiology to manipulate neural circuits implicated in heroin withdrawal and relapse. I will use optogenetics and chemogenetics to determine whether this pathway is necessary and sufficient to drive aversion or hyperalgesia during heroin withdrawal, as well as relapse to heroin seeking. I will validate the functionality of the optogenetic and chemogenetic approaches in vitro using brain slice electrophysiology. My preliminary data indicate that activating the PVT?NAc pathway is sufficient to drive aversion and heroin seeking during acute and extended withdrawal after abstinence from heroin self-administration, but not after extinction training. The PL?NAc pathway has been proposed as a ?final common pathway? to drug seeking and it has been shown to drive heroin seeking after extinction training. The extinction procedure may thus engage the prefrontal cortex to diminish the role of the PVT?NAc pathway in heroin seeking. A central hypothesis of this proposal is that the prelimbic cortex (PL) drives heroin seeking during acute opioid withdrawal and after abstinence through an indirect PL?PVT?NAc pathway, but through a direct PL?NAc pathway after extinction training. During the R00 independent phase of this proposal I will test this hypothesis using chemogenetics to inhibit the PL?PVT pathway to attenuate heroin withdrawal-induced hyperalgesia and relapse. One means by which this shift in circuits may occur, depending on withdrawal modality, is by shifting glutamatergic drive to the same population of NAc neurons that drive heroin seeking. To assess this, I will investigate whether a subset of NAc neurons receives convergent inputs from both the PL and PVT using slice electrophysiology. Another means by which this circuitry may evolve to drive heroin seeking is through changes in synaptic strength within PL?PVT?NAc pathway. I will thus investigate synaptic strength (measured as AMPA/NMDA ratio) within this circuit. Next, I will test the ability of a long-term depression protocol (LTD) in vitro to normalize identified changes in synaptic strength, and then apply the same protocol in vivo to reduce heroin seeking and hyperalgesia during withdrawal from heroin self-administration. These experiments will reveal the role of PVT circuits in heroin withdrawal and how these circuits interface with known relapse circuitry to control heroin seeking.